S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Agricultural Biotechnology (농생명공학부) Theses (Master's Degree_농생명공학부)
Development and characterization of trivalent subunit vaccine for defending various serotypes of Foot-and-Mouth disease viruses
다양한 혈청형의 구제역 바이러스 방어를 위한 3가형 아단위 백신의 개발 및 특성규명
- 농업생명과학대학 농생명공학부
- Issue Date
- 서울대학교 대학원
- Foot-and-mouth disease; subunit vaccine; multi-epitope; trivalent vaccine; chaperone co-expression system; alkaline-pH acetone precipitation; inclusion bodies; solublization
- 학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 최윤재.
- Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals like pig, cow, horse, sheep and goat. It is classified number 1 communicable disease and International Enzootic des Office (OIE) is trying to control its contagion to abroad. Clinical symptoms of FMD are the development of lesions resembling blisters on the feet and around of the mouth, fever, depression and lameness. Adult animals can recover from FMD within two weeks after infection. But, because of high contagion and mutant, slaughter of infected and susceptible animals is came into action and it causes economic losses to livestock industry. The country which is not recognized as FMD-free by OIE is placed restrictions on international trade. DIVA (Differentiation Infected from Vaccinated Animals) problem is a main reason of restriction on international trade. Vaccine used in farm is live attenuated or inactivated FMD vaccine. Because live attenuated or inactivated FMD vaccine contains non structural proteins, it is impossible to distinguish live attenuated or inactivated vaccinated animals from infected animals. In addition, live attenuated or inactivated FMD vaccine cant defend various serotypes and subtypes of FMDV.
To overcome disadvantages of live attenuated or inactivated FMD vaccine, subunit vaccine strategy was introduced. Because producing subunit vaccine is simple, disease control is more faster than live attenuated or inactivated vaccine. By not using non structural protein of FMDV, it is possible to distinguish subunit vaccinated animals from infected animals. But the main problem of subunit vaccine is low immunogenecity. To overcome low immunogenecity of subunit vaccine, we introduced multi-epitope vaccine strategy which is linked epitopes having high antigenecity. Also, to defend various serotypes and subtypes of FMDV, trivalent vaccine strategy was introduced. Serotype O, serotype A and Asia-1s three epitopes of three subtypes were combined, respectively. As a result three vectors were constructed
recombinant protein of serotype O was named O9BT, recombinant protein of serotype A was named A9BT and recombinant protein of Asia-1 was named I9BT.
We used E. coli expression system to produce trivalent multi-epitope subunit vaccine. Multi-epitope subunit vaccine is not a native structure of FMDV but a artificial form of vaccine. The problem of artificial vaccine is that it is expressed as a form of inclusion bodies. Because trivalent multi-epitope subunit vaccine we constructed also expressed as a form of inclusion bodies, we introduced solubilization methods
chaperone co-expression system and alkaline-pH acetone precipitation. Production efficiency of soluble O9BT was the highest among three recombinant proteins, we used O9BT to compare solubilized proteins with soluble proteins. First, chaperone co-expression system was used to induce production of soluble protein by delaying proteins folding or transforming misfolded and aggregated proteins. Because pTf16, which delays protein folding when it is extruded from exit site of ribosome, was the most effective among five types of chaperones plasmids, it was selected for mass production and in vivo immunization. Second, inclusion bodies were solubilized by introducing alkaline-pH acetone precipitation. After solubilization by alkaline buffer (> pH 12.5), cold acetone was added to precipitate proteins. Precipitated proteins can solubilize in PBS or D.W because of protein refolding. Especially alkaline-pH acetone precipitation uses inclusion bodies, so its production efficiency is higher than other production methods. We compared solubilized proteins produced by chaperone co-expression system and alkaline-pH acetone precipitation with soluble proteins through in vivo immunization. Regardless of solubilization methods, soluble proteins induced similar immune responses. Finally, alkaline-pH acetone precipitation was selected for trivalent multi-eptitope subunit vaccine production. Because its production efficiency was 4 times higher than other methods and it didn't need purify step.
Trivalent multi-epitope subunit vaccine (O9BT, A9BT, I9BT) were produced by alkaline-pH acetone precipitation and 20 μg or 45 μg of cocktail trivalent multi-epitope subunit vaccine was injected to mice intramuscularly. Even though amount of each protein were reduced one third, the efficiency of trivalent multi-epitope subunit vaccine corresponded to monovalent multi-epitope subunit vaccine. Expecially, in neutralization assay, only 20 μg of trivalent multi-epitope subunit vaccine was effective. Interestingly, the vaccine efficiency was not necessarily proportional to amount of vaccine, because 20 μg and 45 μg of trivalent multi-epitope subunit vaccine showed similar immune responses.
We have shown through this study that trivalent multi-epitope subunit vaccine can overcome the antigenic variation of FMDV. In order to be used as a practical commercial vaccine, additional experiments such as a neutralizing antibody assay and challenge assay will be necessary. But it is expected that trivalent multi-epitope FMD subunit vaccine can increase immunogenecity of subunit vaccine and effectively defend various serotypes of FMDV in livestock industry.